In microorganisms, biosynthesis of L-arginine proceeds in eight enzymatic steps starting from the precursor L-glutamate and follows two different pathways, the linear pathway or the cyclic acetyl pathway depending on the microorganism concerned (Cunin et al., 1986; Davis, 1986). In both biosynthetic pathways the first step is N-transacetylation of glutamate catalyzed by the enzymes displaying N-acetylglutamate synthase activity.
In the linear pathway, the acetylglutamate synthase activity is provided by the enzyme acetylCoA: L-glutamate N-acetyltransferase (EC 2.3.1.1.) encoded by the argA gene and in this pathway the intermediate N-acetyl L-ornithine is converted into L-ornithine at the fifth enzymatic step through deacetylation by N2-acetyl-L-ornithine amidohydrolase (EC 3.5.1.16) encoded by the argE gene.
Thus, in microorganisms as Escherichia coli, L-arginine is synthesized from L-glutamate via N-acetylglutamate, N-acetylglutamylphosphate, N-acetylglutamate semialdehyde, N-acetylornithine, ornithine, citrulline and argininosuccinate. These intermediates are synthesized through consecutive reactions catalyzed by enzymes commonly known under the names N-acetylglutamate synthase, N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine carbamyltransferase, argininosuccinate synthase and argininosuccinase. These enzymes are encoded by argA, argB, argC, argD, argE, argF, argG and argH genes, respectively.
In the cyclic acetyl pathway, the acetyl-group of N-acetylornithine is transferred to L-glutamate by the enzyme ornithine acetyltransferase (N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase; EC 2.3.1.35) encoded by the argJ gene. Owning this enzyme, arginine biosynthetic pathway is recycled between the first and the fifth enzymatic steps and such a cyclic acetyl pathway is energetically advantageous over the linear pathway since N-acetyl ornithine can be used as the acetyl-group donor once the pathway is initiated from acetyl-CoA as a donor.
The cyclic acetyl pathway directs the L-arginine flow in procaryotic organisms as Corynebacterium glutamicum (Udaka and Kinoshita, 1958), cyanobacteria (Hoare and Hoare, 1966), Pseudomonas aeruginosa (Haas et al., 1972), Neisseria gonorrhoeae (Shinners and Catlin, 1978), methanogenic archaea (Meile and Leisinger, 1984), Thermotoga maritima (Van de Casteele et al., 1990), representatives of Bacillus (Sakanyan et al., 1992), Streptomyces coelicolor (Hindle et al., 1994), Thermus thermophilus (Baetens et al., 1998), an archaeon Methanococcus jannaschii (Marc et al., 2000) and in some eukaryotic organisms (De Deken, 1962). The nucleotide or amino acid sequences sharing similarity with the argJ gene or its product are also available for entirely or partially sequenced genomes and the similarity is indicative of the existence of the cyclic acetyl pathway in these organisms.
The argJ-encoded product, which exhibits the only ornithine acetyltransferase, is considered as a monofunctional enzyme and properties of such enzyme have been described (Haas et al., 1972; Sakanyan et al., 1996; Baetens et al., 1998; Marc et al., 2000). However, some microorganisms harbour the alternative version of the argJ gene encoding the enzyme which possesses, in addition to the ornithine acetyltransferase activity, the N-acetylglutamate synthase activity as well. Such genes and corresponding bifunctional enzymes have been described for Neisseria gonorrhoeae (Picard and Dillon, 1989; Martin and Mulks, 1992), B. stearothermophilus (Sakanyan et al., 1990 and, 1993), Saccharomyces cerevisiae (Crabeel et al., 1997), T. neapolitana (Marc et al., 2000).
The monofunctional ArgJ enzymes can be distinguished from bifunctional enzymes by two means: (i) by enzymatic assay using two acetyl-group donors, N-acetyl L-ornithine and acetyl-CoA; (ii) by complementation test using argE and argA mutants of Escherichia coli for the cloned argJ gene. The monofunctional ArgJ enzyme transfers the only acetyl group from N-acetyl L-ornithine to L-glutamate and complements the only argE mutant, whereas the bifunctional ArgJ enzyme transfers the acetyl-group both from N-acetyl L-ornithine and acetyl-CoA and complements both argE and argA mutant strains.
Both biosynthetic pathways are subjected to genetic and enzymatic regulation, respectively by a specific transcriptional repressor and by inhibition of enzymatic steps by L-arginine or intermediate products (Maas, 1994; Glansdorff, 1996). Moreover, the early metabolic steps preceding the L-glutamate precursor formation and late degradation steps following the L-arginine degradation are under the control of regulatory mechanisms. Consequently, synthesis of L-arginine and the production yield of this amino acid can be modulated by introduction of mutations at various targets in the genome of a given microorganism or by affecting the cultivation conditions of a given microorganism or by affecting the membrane permeability of a given microorganism.
Conventional L-arginine production by fermentation has been carried out using microbial strains producing L-arginine, especially representatives of coryneform bacteria; using coryneform bacteria resistant to certain antimetabolic agents including 2-thioazoalanine, α-amino-β-hydroxyvaleric acid, arginine hydroxamate, cysteine analogues, sulfonamide derivatives and the like; using coryneform bacteria exhibiting auxotrophy for some amino acids including for L-proline, L-histidine, L-threonine, L-isoleucine, L-methionine, or L-tryptophan, as well as using coryneform bacteria exhibiting both the mentioned above resistances and auxotrophies for amino acids. In this respect, reference may be made to the following patents: FR 2 084 059, 2 119 755, 2 490 674, 2 341 648, 2 225 519, EP 0 379903 B1, EP 0 378 223 B1, EP 0 336387 B1.
On the other hand, there have been disclosed methods for producing L-arginine by using a microorganism belonging to the genus Corynebacterium, Brevibacterium or Escherichia transformed by a recombinant DNA containing a well-defined gene of arginine biosynthesis that allows to enhance the gene-encoded enzyme activity for a given limiting step. The wild-type strain or the mutant for the transcriptional repressor or the mutant which carries a relevant resistance or auxotrophy have been used as recombinant host cell for fermentations.
Most of the recombinant microorganisms used for producing L-arginine belong to the genus Corynebacterium or Brevibacterium. In this respect, reference may be made to the following patents: FR 2 143 238; FR 2 484 448; EP 0 259858 B1; EP 0 261627 B1; EP 0 332233 A1; EP 0 999267 A1; EP 1016710 A2.
However, the Escherichia coli K12 strain, with the entirely sequenced genome (Blattner et al., 1997) and applicability of various genetic approaches and more advantageous vectors to manipulate in this strain or its derivatives, is an attractive host as well for the production of amino acids including L-arginine. The increased production of L-arginine by recombinant Escherichia coli strains can be achieved by using the cloned argA gene on plasmid vectors and followed by isolation of feed-back resistant mutations by the described method for E. coli (Eckhard and Leisinger, 1975; Rajagopal et al., 1998). In this respect, reference may be made to EP 1 016 710 A2.
Thus, L-arginine production by recombinant microorganisms has been improved by enhancing the number of copies of the gene coding for N-acetylglutamate synthase activity, namely by a wild type argA gene or its feedback resistant mutants.
However, the application of the mutant argA gene is limited in the context of a possibility of further increasing the productivity of L-arginine by recombinant strains.
It has now been found that it is possible to produce L-arginine with a microorganism having an L-arginine producing ability, said microorganism being a microorganism synthesizing L-arginine and bearing a recombinant DNA comprising a gene argJ coding an enzyme with an ornithine acetyltransferase activity.